Process for preparing phosphoric acid monoesters of mannose polymers



3,002,967 PROCESS FOR PREPARING PHOSPHORIC ACID MCNOESTERS F MANNOSE POLYMERS Morey E. Slodki, Peoria, 11L, assignor to the United States of America as represented by the Secretary of Agriculture No Drawing. Filed Dec. 18, 1959, Ser. No. 860,615 7 2 Claims. (Cl. 260-234) (Granted under Title 35, U.S. Code (1952), see. 266) phomannan polymers, namely the phosphomannan poly-- mers produced in the aerobic whole culture fermentations of Hansenula holstii, NRRL Y-2448, Y-2154, and Y- 2155, as disclosed and claimed in Patent No. 2,961,378 of Benedict et al., and also the phosphoric acid monoesters derived from the potassium salts of the phosphomannan polymers produced. in corresponding fermentations of Hansenula capsulata, NRRL Y-1889, H. capsulata, NRRL Y- 1842, H. hostii, NRRL Y-2154, H. minuta, NRRL Y 411, Pachysolen tannophilus, NRRL, Y-246l, Torulopsz's pinus, NRRL Y- -202 3, and Szzccharomyces pini, NRRL YB-2022.

The phosphoric acid monoesters prepared from the potassium salts ofithe above polymers possess marked dis.- persant action so that, for example, a very small amount admixed with 100 gm. of zinc oxide in 100 ml. of water provides a smooth fluid dispersed paste instead of an extremely thick lumpy paste. Such free-flowing pastes can advantageously be employed in skin lotions and ointments. It is emphasized that the action of my monoesters is not a suspending action as would be provided by a gum-like polymer, and that aqueous solutions thereof are clear, water-white, and tasteless, the 1.0 percent aqueous solutions having a viscosity of about 1.5 centistokes at 25 C.

I have discovered that the potassium phosphomannans obtained by methanol precipitation from the said fermentations are converted to the pyrophosphoric acid form of the phosphomannan by slurrying with the hydrogen form of a sulfonic acid type cation exchange resin. The viscosity of the somewhat viscous solution falls abruptly and the pyrophosphoric acid form of the polymer can simply be decanted 01f, leaving the cation exchange resin. Moderate heating of the decanted pyrophosphoric acid form causes an autohydrolysis and the formation of the phosphoric acid form without significant concomitant hydrolysis of the mannose polymer chains.

The complete structures of the autohydrolyzed phosphomannan polymers of this invention have not yet been fully established, but since over 95 percent of the phosphorus content has been isolated in the form of mannosefi-phosphoric acid as shown in my concurrently filed application, Serial No. 860,614, the phosphoric acid moiety is bound to the No. 6 carbon atoms of the mannosidic units.

Table I shows the degrees of polymerization and indicates the extent of phosphorylation of the potassium phosphomannan starting materials. It permits selection of the most advantageous member for a given purpose. TABLE I Yeast phosphomzmnans Molar 1 Organism NRRL ratio of D.P.

No. mannose to phosphorus Hansemila capsulata... Y-1889 2. 5 101 Do Y-l842 2. 5 244 Pachysolen tannophilua Y-2461 4. 2 256' Y-2l54 .5. 2 v600 D0 Y-2448 5. 7 588 Torulopsis ptnu Y-2033 8. 4 51 Sac. pini YB-2022 12. 2 51' H. minute Y-411 27. 5 144 Table II shows the dispersant efiect on zinc oxide of adding 1 percenttbased on the zinc oxide) of neutralized autohydrolyzed Y-l'842 and Y-2448 phosphomannanw The following mixtures were prepared:

A. 30 gm. ZnO powder+30 ml. HOH. B. 30 gm. ZnO powder+30 ml. HOH+1% NaCl C. 30 gm. ZnO powder+30 ml. 1% autohyd. Y-1-S42 P-M- D. gm. ZnO powder+30 m1. 1% autohyd. Y2448 P-M -.T ABLE II [Bro0kfie1d v1sc0s1meter, N o. 3 spindle] 30 Sample 7 g 6 r.p.m. 12 rpm. 30 r.p.m. 60 r.p.m. 1co, 000 i I 1 Too viscous to read even at lowest r.p.m.

Phosphomannan, NRRL Y-1842, in the form of its potassium salt, was prepared by culturing 200 ml. of a freshly prepared subculture of Hansenula capsulata, NRRL Y-1842, for 96 hours at 25 C. under aerobic conditions (0.5 millimole O /L/min.) with 19 volumes of a substrate having the following composition:

Component: Wt. by vol., percent Comml. glucose 6.0 Corn steep liquor 0.1 Tryptone 0.1 KH PO 0.5 Solution B (Speakman salts), v./v 0.5 Water, q.s.

(Initial pH, 5.0.) 0. Biol. Chem. 58, 395 (1923-1924:).

Phosphomannan, NRRL Y-1842, in the form of its potassium salt was recovered by supercentrifuging otf the yeast cells after adding 2100 ml. methanol (providing a 50 percent concentration); adding 30 ml. of saturated potassium acetate solution to precipitate the potassium salt of said phosphomannan from the centrifugate; redissolving the potassium salt of the phosphomannan in 6000 m1. of SO-percent aqueous methanol; recentrifuging; reprecipitating the potassium salt of the phosphomannan from the supernatant by adding 40 ml. of saturated potassium acetate solution; redissolving the potassium phosphomannan in 1500 ml. distilled water; adding 900 ml. of methanol containing percent potassium acetate (final methanol concentration of 37 percent); dehydrating the precipitated potassium phosphomannan, NRRL Y-1842, in 600 m1. methanol containing 1 percent potassium acetate; washing with absolute methanol; and drying in a vacuum oven for 8 hours at 45 C. Yield: 55 grams of pure white powder.

Two grams of potassium phosphomannan, NRRL Y- 1842, obtained as above was dissolved in 200 ml. distilled water and slurried with an excess (until Congo red turned blue) of the hydrogen form (H''') of a sulfonic acid type cation exchange resin. The viscosity of the phosphomannan solution fell abruptly so that the cation exchange resin settled and permitted simple decantation .of the solution containing the pyrophosphoric acid form of the phosphomannan. Heating of the decanted solution for 20 minutes at 100 C. provided autohydrolysis of the pyrophosphoric acid group and formation of the phosphoric acid monoester form of phosphomannan, NRRL Y-1842. The 1 percent solution thusly prepared was employed after neutralization in the zinc oxide dispersion studies evaluated in Table I, but it is obvious that for commercial purposes it might be advantageous to provide the neutralized dry product or partially evaporated solutions.

Having fully disclosed my invention, I claim:

1. A method of producing the phosphoric acid monoester of a phosphomannan polymer obtained from the aerobic whole culture fermentation of a yeast selected from the group consisting of Hansenula holstii NRRL Y-2448, Hansenula holstii NRRL Y-2154, Hansenula holstii NRRL Y-2155, Hansenula capsulata NRRL Y- 1889, Hansenula capsulata NRRL Y1842, Hansenula minuta NRRL Y-411, Pachysolen tarmophilus NRRL Y- 2461, Torulopsis pinus NRRL Y-2023, and Sarcharomyces pim' NRRL YB-2022, comprising slurrying said phosphomannan polymer in the form of its potassium salt with the hydrogen form of a sulfonic acid type cation exchange resin to form a solution containing the pyrophosphoric acid form of said phosphomannan polymer, separating said solution from the slurry, and heating said solution at a temperature of about C. for about 20 minutes to effect autohydrolysis of the pryophosphoric acid group and formation of the phosphoric acid monoester form of said phosphomannan polymer.

2. A method of producing the phosphoric acid monoester of a phosphomannan polymer obtained from the aerobic whole culture fermentation of a yeast selected from the group consisting of Hansenula holstii NRRL Y-2448, Hansenula holstii NRRL Y-2l54, Hansenula holstii NRRL Y-2155, Hansenula capsulata NRRL Y 1889, Hansenula capsulata NRRL Y-l842, Hansenula minuta NRRL Y-411, Pachysolen tannophilus NRRL Y- 2461, Torulopsis pinus NRRL Y-2023, and Saccharomyces pini NRRL YB-2022, comprising slurrying said phosphomannan polymer in the form of its potassium salt with the hydrogen form of a sulfonic acid type cation exchange resin to form a solution containing the pyrophosphoric acid form of said phosphomannan polymer, separating said solution from the slurry, heating said solution at a temperature of about 100 C. for about 20 minutes to efiect autohydrolysis of the pyrophosphoric acid group and formation of the phosphoric acid monoester form of said phosphomannan polymer, and neutralizing the resulting solution.

References Cited in the file of this patent UNITED STATES PATENTS 1,129,321 Von-Euler-Chelpin Feb. 23, 1915 2,023,036 Funaoka Dec. 10, 1935 2,052,029 Harris Aug. 25, 1936 2,520,980 Terkel Sept. 5, 1950 2,678,902 Mehafiey May 18, 1954 

1. A METHOD OF PRODUCING THE PHOSPHORIC ACID MONOESTER OF A PHOSPHOMANNAN POLYMER OBTAINED FROM THE AEROBIC WHOLE CULTURE FREMENTATION OF A YEAST SELECTED FROM THE GROUP CONSISTING OF HANSENULA HOLSTII NRRL Y-2448, HANSENULA HOLSTII NRRL Y-2154, HANSENULA HOLSTII NRRL Y-2155, HANSENULA CAPSULATA NRRL Y1889, HANSENULA CAPSULATA NRRL Y-1842, HANSENULA MINUTA NRRL Y-411, PACHYSOLEN TANNOPHILUS NRRL Y2461, TORULOPSIS PINUS NRRL Y-2023, AND SACCHAROMYCES PINI NRRL YB-2022, COMPRISING SLURRYING SAID PHOSPHOMANNAN POLYMER IN THE FORM OF ITS POTASSIUM SALT WITH THE HYDROGEN FORM OF A SULFONIC ACID TYPE CATION EXCHANGE RESIN TO FORM A SOLUTION CONTAINING THE PYROPHOSPHORIC ACID FORM OF SAID PHOSPHOMANNAN POLYMER, SEPARATING SAID SOLUTION FROM THE SLURRY, AND HEATING SAID SOLUTION AT A TEMPERATURE OF ABOUT 100*C. FOR ABOUT 20 MINUTES TO EFFECT AUTOHYDROLYSIS OF THE PRYOPHOSPHORIC ACID GROUP AND FORMATION OF THE PHOSPHORIC ACIC MONOESTER FORM OF SAID PHOSPHOMANNAN POLYMER. 